The oxidation status of HDL plays an important role in determining how this lipoprotein prevents or promotes atherosclerosis. Since patients with normal levels of HDL experience atherogenic events (ie., stroke, myocardial infarction), HDL function itself may be a stronger indicator of cardiovascular disease than the actual levels of HDL cholesterol. Bioassays of HDL function are complex, time-consuming, technician- and technique-dependent and difficult to reproduce between labs. Thus, highly efficient assays of HDL function are desperately needed. In this application, we hypothesize that the biophysics of HDL interactions with biomolecules the mediate HDL-dependent cholesterol metabolism represent a novel source of physiological information that can be used to assess the functional state of HDL. Using biolayer interferometry (BLI), a new label-free technique for measuring biomolecular interactions, we will develop a series of novel assays of HDL function to determine if and the extent to which oxidation impacts on the ability of HDL to interact with 1) anti- and pro-inflammatory enzymes (paraoxonase [PON1], platelet activating factor acetylhydrolase [PAF-AH], myeloperoxidase [MPO] and xanthine oxidase [XO]) to prevent LDL oxidation; 2) or ATP binding cassette (ABC) transporter 1 (ABCA1)/ABCG1, lecithin cholesteryl acyl transferase (LCAT), cholesteryl ester transfer protein (CETP) and scavenger receptor class B1 (SR-B1) to mediate HDL-cholesterol release, esterification, transfer and uptake, respectively. The mechanisms by which HDL is anti-inflammatory or participates in reverse cholesterol transport is directly dependent on the ability of HDL to bind these biomolecules. As such, any oxidation-induced changes in HDL binding affinity for these biomolecules should provide a sensitive index of HDL functionality. In Aim 1, we propose to measure the extent to which oxidized lipids and proteins in reconstituted HDL (r-HDL) impair HDL function using established in vitro bioassays of cholesterol release, esterification, transfer and uptake. In Aim 2, we will ue BLI assays to determine how oxidized lipids and proteins in r-HDL alter binding rates and affinity for the proteins and enzymes that mediate HDL's ability to inhibit LDL oxidation or promote specific steps in the reverse cholesterol transport pathway. In Aim 3, we propose to verify and validate that BLI assays by 1) correlating BLI assays with bioassays of HDL function in established murine models of vascular disease and 2) by using BLI assays to predict which patients have clinically-diagnosed atherosclerosis in case-controlled, blinded human studies. Successful completion of these studies will lay the foundation for the development of a new clinical assay for determining HDL functionality. Validation of these new assays will allow us to identify which patients have dysfunctional HDL in a time frame and for a cost that is compatible with clinical reference laboratories. Development of these novel assays will make it possible, for the first time, to perform large clinical studies to fully test the idea that dysfunctional HDL is better indicator of atherosclerotic risk.